The proposal describes a series of experiments designed to target and track antigen following oral delivery via edible transgenic plants. The overall objective is to enhance both systemic and mucosal immune responses to orally delivered antigen. During the previous grant period we have demonstrated that mice fed potatoes expressing HBsAg make both primary and secondary antibodies responses in serum and anti-HBs IgA responses in mucosal secretions. We have provided transmission electron microscopic evidence that the transgenic potato cells harbored unusual membrane-bound bodies that contained circular structures that are approximately 17 nm in diameter. These structures are very similar to the virus-like particles (VLP) found in the serum samples of HBV infected patients and the distended ER vesicles are similar to those produced in yeast expressing HBsAg. These images provide the first ever evidence that in transgenic plants antigen is refined within vesicular structures. We also performed a successful double-blind placebo controlled Phase 1 trial to evaluate the safety and immunogenicity of the edible plant vaccine. None of the volunteers who ate nontransgenic potatoes showed changes in anti-HBs antibody titer. However, after volunteers ate transgenic potatoes, serum antibody titers increased in 62% who ate three doses and 53% who ate two doses of transgenic potatoes. Buffering of the stomach pH was not performed nor was any mucosal adjuvant used. This trial firmly establishes that oral vaccination via edible plant vaccines is possible for a non-enteric antigen such as HBsAg. Taken together, these data form a very solid foundation for the studies proposed in this application. In the current application we propose four specific aims designed (1) to improve the expression of HBsAg in transgenic plants, (2) target antigen to M cells in the gut, (3) to track the immune response elicited and (4) to potentiate the immune response to orally administered antigen by use of nontoxic mucosal adjuvants. We have provided significant new preliminary data and we believe that these provide strong documentation of the likelihood of continued success.